Acute myeloid leukemia (AML) is the most prevalent form of leukemia, resulting from oncogenic transformation of the myeloid progenitor cells. Mutation of Ras proteins to a continuously activated state predominates in cancer, including AML. Chronic Ras activation increases cell division, a hallmark of cancer. Ras GTPases cycle from an active GTP-loaded state to an inactive GDP-loaded state. We have discovered that the nucleotide-free transition-state of Ras formed during the exchange of GTP for GDP can negatively regulate protein targets, adding a new dimension to Ras signaling. There are three members of the Ras sub- family; KRas, HRas and NRas, each mutated in a distinct subset of cancers. Activated KRas is a common driving force in cancer. A second class of proteins, called PI3K's, is known for activating the AKT pathway that prevents pre-programed cell death, another hallmark of cancer. This project focuses on a class II PI3K called PI3KC2?, which is overexpressed in several cancers, including AML and neuroblastoma. It has been shown that inhibition of PI3KC2? leads decreased proliferation in AML cell lines. We have found that HRas, in its nucleotide free state, interacts with PI3KC2? inhibiting its activation in vitro. In contrat, my preliminary data shows that activated KRas (GTP-KRas) interacts with PI3KC2?, suggesting that the KRas:PI3KC2? complex is active. We have previously performed in vitro GST pulldowns to show that HRas, in the absence of nucleotide preferentially interacts with PI3KC2? and that this interaction is bimolecular. I will use this approach to test whether GTP-loaded KRas interacts with PI3KC2?. My first aim will be to define the sequence of HRas and KRas, which provides for such nucleotide-dependent interaction with PI3KC2?. The nucleotide free HRas:PI3KC2? interaction occurs on intracellular vesicles whereas activated KRas interacts with PI3KC2? on the plasma membrane. In this project I will switch the regions required for Ras localization between HRas and KRas to determine the requirements for PI3KC2? localization. My second aim will determine the activation state of PI3KC2? in cells during both of these interactions, along with the ability of HRas vs KRas to sequester PI3KC2?. These experiments will be achieved by transfecting fluorescent probes specific for PI3KC2?'s lipid product PI(3,4)P2 (an AKT activator) into cells. This allows visualization of various lipid products using these genetically encoded probes and their co-localization with fluorescently tagged PI3KC2?. The co-transfection of HRas and/or KRas will determine the effects of each Ras isoform on PI3KC2? activity. My final aim utilizes AML cell lines where KRas is either wildtype or activated and determines the effects of modulating wild type HRas levels on proliferation and resistance to apoptosis and whether activated KRas negates this effect. Conversely, I will silence HRas (therefore preventing PI3KC2? inhibition by HRas) on the tumorigenic characteristics of AML lines. This research provides additional evidence that Ras can negatively regulate protein targets and that different Ras isoforms signal differently through the same target.